Micro-CT imaging of Onchocerca infection of Simulium damnosum s.l. blackflies and comparison of the peritrophic membrane thickness of forest and savannah flies.

Onchocerciasis is a neglected tropical disease (NTD) caused by Onchocerca Diesing 1841 (Spirurida: Onchocercidae) nematodes transmitted by blackflies. It is associated with poverty and imposes a significant health, welfare and economic burden on many tropical countries. Current methods to visualize infections within the vectors rely on invasive methods. However, using micro-computed tomography techniques, without interference from physical tissue manipulation, we visualized in three dimensions for the first time an L1 larva of an Onchocerca species within the thoracic musculature of a blackfly, Simulium damnosum s.l. Theobald 1903 (Diptera: Simuliidae), naturally infected in Ghana. The possibility that thicker peritrophic membranes in savannah flies could account for their lower parasite loads was not supported, but there were limits to our analysis. While there were no statistically significant differences between the mean thicknesses of the peritrophic membranes, in the anterior, dorsal and ventral regions, of forest and savannah blackflies killed 34-48 min after a blood-meal, the thickness of the peritrophic membrane in the posterior region could not be measured. Micro-computed tomography has the potential to provide novel information on many other parasite/vector systems and impactful images for public engagement in health education.

Major differences in the larval anatomy of the digestive and excretory systems of three Oestridae species revealed by micro-CT.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro-computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a 'glandular band' by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio-opaque concretions. Moreover, the anatomy of O. ovis third-instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a 'band' and anterior Malpighian tubules narrowly uniform throughout their entire length.

Internal morphological changes during metamorphosis in the sheep nasal bot fly, Oestrus ovis.

During the larval stage, oestrid flies (Diptera: Oestridae) are obligate parasites, whereas during the adult stage they are free-living and do not feed. Like other cyclorrhaphous flies, oestrids undergo metamorphosis inside an opaque puparium, formed by the contracted and hardened cuticle of the third-instar larva. The present study documents the internal morphological changes taking place during metamorphosis of the sheep nasal bot fly, Oestrus ovis L., using non-invasive, micro-CT-based virtual histology and provides quantitative data of volumetric changes in specific organs. Virtual histological sections allowed visualisation of the progression and completion of the apolyses, which delimit the different intra-puparial stages, and the connection to the tracheal system of a large gas bubble, which plays an essential role during early metamorphosis. Overall, our results show that the sequence of morphological and volumetric changes in tissues and organs is similar to those found in other cyclorrhaphous flies, but they also reveal developmental differences that result in an adult vestigial digestive tract. Future studies could develop non-invasive, reliable methods for aging the intra-puparial forms of different oestrid species of veterinary importance, based on both qualitative and quantitative markers, thus improving our knowledge of their development and the efficiency of control strategies.

Micro-computed tomography visualization of the vestigial alimentary canal in adult oestrid flies.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage as they acquire all necessary nutrients during the parasitic larval stage. The adult mouthparts and digestive tract are therefore frequently vestigial; however, morphological data on the alimentary canal in adult oestrid flies are scarce and a proper visualization of this organ system within the adult body is lacking. The present work visualizes the morphology of the alimentary canal in adults of two oestrid species, Oestrus ovis L. and Hypoderma lineatum (de Villiers), with the use of non-invasive micro-computed tomography (micro-CT) and compares it with the highly developed alimentary canal of the blow fly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae). Both O. ovis and H. lineatum adults showed significant reductions of the cardia and the diameter of the digestive tract, an absence of the helicoidal portion of the midgut typical of other cyclorrhaphous flies, and a lack of crop and salivary glands. Given the current interest in the alimentary canal in adult dipterans in biomedical and developmental biology studies, further understanding of the morphology and development of this organ system in adult oestrids may provide valuable new insights in several areas of research.

Sarcosaprophagous Diptera assemblages in natural habitats in central Spain: spatial and seasonal changes in composition.

The composition and spatial distribution of sarcosaprophagous Diptera assemblages were studied using carrion-baited traps along a bioclimatic gradient of natural habitats in central Spain throughout the different seasons during 1 year. Calliphoridae and Muscidae were the most abundant families, accounting for, respectively, 41.9% and 35.1% of all Diptera specimens collected. Other abundant families were Heleomyzidae (8.4%), Sarcophagidae (6.9%) and Piophilidae (5.1%). Fly assemblage compositions differed among bioclimatic levels, with Chrysomya albiceps (Wiedemann) (Diptera: Calliphoridae) being the dominant species in mesomediterranean habitats, Muscina levida (Harris) (Diptera: Muscidae) the dominant species in supramediterranean habitats, and Prochyliza nigrimana (Meigen) (Diptera: Piophilidae) the dominant species in oromediterranean habitats. Differences in assemblage composition were also found among seasons. Thermophobic species such as Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) and some species of Heleomyzidae were well represented during autumn, winter and spring in the three bioclimatic levels sampled. By contrast, thermophilic species such as Ch. albiceps and Lucilia sericata (Meigen) (Diptera: Calliphoridae) and most Muscidae and Sarcophagidae species were more abundant during summer and in mesomediterranean habitats located at lower elevations. Knowledge of the preferences of some species for certain habitats may be of ecological and forensic value and may establish a starting point for further research.

New piophilid in town: the first Palaearctic record of Piophila megastigmata and its coexistence with Piophila casei in central Spain.

The genus Piophila Fallén (Diptera: Piophilidae) is known from only two species: Piophila casei (L.) is a major pest, a cosmopolitan species and is commonly used as a forensic indicator, whereas Piophila megastigmata McAlpine has until now only been recorded in a natural environment in South Africa. The present work reports the first occurrence of P. megastigmata in the Palaearctic region from specimens collected by carrion-baited traps throughout different natural habitats of central Spain. Furthermore, the species was also collected with P. casei on corpses of domestic pigs used in a carrion succession study in a periurban habitat in central Spain. Both species occurred on carrion in different seasons, but P. megastigmata was more abundant than P. casei in autumn, arriving earlier at the carcasses and persisting for a longer period. The contrary pattern was observed in spring. The presence of P. megastigmata in different localities in central Spain and its coexistence with P. casei in a periurban habitat make it a potentially useful new tool for legal medicine in Europe; thus this species must be considered in forensic entomology studies.

La Entomología Forense en España: pasado, presente y perspectivas de futuro / Forensic Entomology in Spain: past, present and future perspectives

La entomología forense -estudio de los insectos que aparecen en los cadáveres- es una disciplina relativamente joven en cuanto a su aplicación. En España ha evolucionado lentamente, experimentando un notable crecimiento en las últimas décadas, tanto en el número de trabajos como en el de grupos de investigación dedicados a ello, adquiriendo un estatus cada vez más importante como disciplina forense. En este trabajo se presentan los resultados de una revisión bibliográfica en la que se ha analizado con detalle la evolución de las publicaciones relacionadas con la entomología forense en España. Se incluye además un anexo con las referencias de todos los artículos recopilados, clasificados según sus características (divulgación científica, educación o formación, aplicación en casos reales e investigación en entomología forense). Finalmente se analiza el estado actual de esta disciplina en nuestro país, considerando la faceta educativa, así como los principales lugares donde se investiga y aquellos donde se aplica la entomología forense en la resolución de casos reales

Forensic Entomology- the study of insects appearing in corpses- is a relatively young discipline regarding its application. In Spain it has evolved slowly showing a great development in the last decades, both regarding the publications number and the people involved, gaining a higher status as a forensic discipline. The results of a bibliographical revision in which evolution of the number of Spanish Forensic Entomology publications has been analyzed in detail are presented. Moreover, an annexe with the references of all the compiled publications classified according to the type (scientific popularization, education and training, application in real cases and investigation in Forensic Entomology) is enclosed. Finally, the current state of the discipline in our country is also presented, taking into consideration education aspects as well as main places where there are people researching or applying forensic entomology in the resolution of real cases